The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blander, J. M.
Right arrow Articles by Janeway, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blander, J. M.
Right arrow Articles by Janeway, C. A., Jr.
The Journal of Immunology, 2003, 170: 2940-2948.
Copyright © 2003 by The American Association of Immunologists

A Pool of Central Memory-Like CD4 T Cells Contains Effector Memory Precursors

J. Magarian Blander*, Derek B. Sant’Angelo{dagger}, Daniela Metz{ddagger}, Sang-Won Kim*, Richard A. Flavell*, Kim Bottomly* and Charles A. Janeway, Jr.1,*

* Section of Immunobiology, Yale University School of Medicine and Howard Hughes Medical Institute, New Haven, CT 06520; and {dagger} Laboratory of T Cell Immunobiology, Memorial Sloan-Ketttering Cancer Center, and Weil Graduate School of Medical Sciences of Cornell University, New York, NY 10021; and {ddagger} David Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, University of Rochester, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The L51S mutation in the D10.G4.1 TCR {alpha}-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the {beta}-chain of I-Ak, and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44highCD62LnegCD45RBneg CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R{alpha} only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Secondary lymphoid tissues contain two main classes of mature CD4 T cells: naive CD4 T cells and memory CD4 T cells. In mice, the classification into one subset or the other is based on several criteria (reviewed in Refs.1, 2, 3, 4, 5). The first criterion is based solely on phenotypic differences. Naive CD4 T cells do not express CD44, but express CD45RB and CD62L, whereas memory CD4 T cells express high levels of CD44 and lower their expression of CD45RB and CD62L. The second criterion is based on functional differences. The first of these relates to the kinetics and efficiency of the response. Memory T cells are thought to be faster and more effective responders to infection than naive T cells. This may be related in part to the higher frequency of circulating Ag-specific memory T cells. At the single-cell level, however, memory T cells have been shown to respond to lower Ag doses and to be less dependent on costimulation for an optimal response. The second functional criterion that differentiates memory cells from naive cells is the pattern of cytokine secretion. Memory T cells produce a variety of cytokines, including the effector cytokines IL-4 and IFN-{gamma}, whereas naive CD4 T cells produce mainly IL-2. The third functional criterion is related to their migration patterns in vivo. Naive T cells recirculate through lymphoid tissues via high endothelial venules (HEVs),2 whereas memory T cells, being L-selectin-negative, cannot use HEVs for entry into lymphoid tissues, instead entering lymph nodes (LN) via afferent lymphatics that drain lymph from peripheral tissues. Memory T cells are thought to be normally present in the blood circulation and, unlike naive T cells, they can extravasate through inflamed endothelium and into noninflamed tissues such as the lungs, liver, gut, and salivary glands (6), where they can execute their effector functions. A final property of memory T cells in this respect is that upon reexposure to Ag, they specifically home to the site where the initial Ag exposure had taken place. This tissue-specific migration, a property that is not shared by naive T cells, is mediated through integrins binding to vascular addressins.

The above criteria used for distinguishing between naive and memory T cells seem straightforward enough. However, because memory T cells arise from naive T cells following antigenic exposure, the distinction between the immediate activated effector cell and the subsequent memory T cell is not always so clear. Activated effector T cells also lower their expression of CD62L and increase their levels of CD44. Nevertheless, one can distinguish activated effector T cells that express CD69 and CD25 (IL-2R), while resting memory T cells do not (4). Therefore, classifying a mature CD4 T cell as a memory cell requires that it possess all these cell surface and functional criteria.

Memory T cells, however, are not a homogeneous population. Recent studies have shown that there are at least two subsets of memory CD4 and CD8 T cells. In humans, these have been termed central memory T cells (TCM) and effector memory T cells (TEM), and although both subsets are CD45RO+, they differ in their expression of the LN-homing receptors CCR7 and L-selectin (7). Two subsets of memory CD4 T cells have also been described in mice (6, 8). As CD4 TCM cells are CCR7+L-selectin+ they recirculate primarily through the LN, whereas TEM cells are CCR7-L-selectin- and recirculate through nonlymphoid tissues (6). The two subsets also appear to have different effector functions; TEM rapidly produce IFN-{gamma} or IL-4, whereas TCM cells produce primarily IL-2 and produce effector cytokines only after a secondary challenge, possibly serving as a new source of effector T cells in vivo.

A further complication to the criteria used for classifying T cells into either naive or memory cells has been solid evidence showing that the CD44highCD45RBlow memory phenotype can also be acquired by T cells that are undergoing homeostasis-driven proliferation. Homeostasis-driven proliferation occurs when naive T cells are confronted with "space" in the periphery, and they proliferate to self-peptide:self MHC ligands to repopulate the T cell compartment (9, 10, 11). Most of our understanding of homeostatic proliferation comes from studies in which a small number of T cells are transferred into immunodeficient hosts that are SCID mice, are recombinase-activating gene deficient, were intentionally irradiated, or were T cell depleted (12, 13, 14, 15). Several elements of homeostatic proliferation were elucidated. First, naive T cells were capable of homeostatic proliferation, resulting in their subsequent masquerade as memory-like T cells. Second, this proliferation in the periphery was independent of thymic output, allowing for the possibility that as the animal ages, and the thymus involutes, the existing naive T cell population may proliferate to prevent the inevitable and harmful decrease in T cell numbers in the periphery. Third, low affinity engagement of the TCR with self MHC molecules loaded with self peptides is necessary. This was evident from studies in which CD8 and CD4 T cells were adoptively transferred into MHC class I- or MHC class II-deficient hosts, respectively (10, 16, 17, 18, 19, 20). In addition, the nature of the peptide itself seemed to be important, since the same sets of self-peptides that induce positive selection in the thymus drive homeostatic proliferation in the periphery (10, 16, 17, 21, 22, 23). Fourth, the cytokine IL-7 is critical for homeostatic proliferation of both naive CD4 and CD8 T cells, as demonstrated by minimal proliferation in IL-7-/- hosts (24, 25, 26) or upon blocking IL-7R with IL-7R{alpha}-specific mAb (27, 28).

Here, we study a previously described TCR transgenic (TCR-Tg) CD4 T cell line, L51S, that was created based on the TCR of the CD4 T cell line D10.G4.1 (D10) (29). Both D10 and L51S T cells recognize, albeit with different affinities, a 13-aa peptide sequence derived from the chicken conalbumin protein (CA134–146) and presented by the MHC class II molecule I-Ak. L51S TCR-Tg mice bear a single mutation at position 51 of the TCR {alpha}-chain CDR2 from L to S that significantly lowers the affinity of TCR to the CA134–146 peptide. Both D10 and L51S mice were bred onto a C{alpha}-/- background to ensure that no endogenous {alpha}-chains were used for pairing with the transgenic {beta}-chain. In separate, but related, studies we now show that this mutation in the TCR that lowers its affinity to its specific MHC:peptide ligand reduces positive selection of immature T cells in the thymus (42) and results in the appearance of mature memory phenotype CD4 T cells in the periphery. In this study we show that the memory phenotype of these CD4 T cells does not translate into effector memory function. Memory phenotype L51S CD4 T cells proliferate in response to lower doses of peptide than naive L51S CD4 T cells, but do not behave like bona fide memory CD4 T cells derived from either syngeneic mice or older D10 TCR-Tg mice. These cells lack immediate effector function, as they do not secrete effector cytokines upon initial TCR stimulation in vitro, but do so more efficiently than naive cells after a second TCR stimulation. This behavior is reminiscent of TCM cells. However, the lack of CCR7 and CD62L expression by these cells, which are markers of TCM cells, precludes us from classifying them as such. We propose that these cells are like TCM cells in that they have a low activation threshold and upon restimulation differentiate into TEM cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic mice

The D10 TCR-Tg mice were generated by microinjection of the rearranged genes encoding the D10.G4.1 {alpha}- and {beta}-chain TCR as previously described (30). The L51S TCR-Tg mice were previously described (29) and were generated by microinjection of the L51S {alpha}-chain containing the L to S mutation at position 51. The L51S TCR-Tg founder was backcrossed to B10.BR and further backcrossed to single D10 {beta}-chain Tg mice (D10 {beta}-chain Tg mice described in Ref.30). Both D10 and L51S TCR-Tg mice were further backcrossed to C{alpha}-/- 129S mice. Homozygous C{alpha}-/- either carrying the transgenes or not were selected for further breeding. The lines were maintained by breeding transgenic C{alpha}-/- mice with nontransgenic C{alpha}-/- mice. The TCR-Tg mice were screened by PCR of tail DNA for the presence of the transgene using D10 TCR {alpha}- and {beta}-chain-specific primers. For the {alpha}-chain, the forward primer sequence was 5' of the ATG, and the reverse primer sequence was 3' of J{alpha} within the intron between J{alpha} and C{alpha}. The sequences were as follows: D10 V{alpha} forward primer, 5'-TTTCTCCCAAACTTCAGTCTA-3'; and D10 V{alpha} reverse primer, 5'-GCTCTGGTCATTGGCACGAT-3'. For the {beta}-chain, the forward primer sequence was within the leader, and the reverse primer sequence was 3' of J{beta} within the intron between J{beta} and C{beta}. The sequences were as follows: D10 V{beta} forward primer, 5'-GCATTCTAGATGGTCCCAAGATGGGC-3'; and D10 V{beta} reverse primer, 5'-TTAAGGATCCACTCTGCTAAGGTTTTCTGC-3'. PCR conditions were 40 s at 94°C, 40 s at 55°C, and 1 min at 72°C for 40 amplification cycles.

Peptides

Peptides were synthesized and purified by the W. M. Keck Biotechnology Resource Laboratory at Yale School of Medicine (New Haven, CT) using tBOC chemistry. Peptides were characterized by reverse phase HPLC, amino acid analysis, and Fab mass spectroscopy. The sequence of peptide CA134–146 is HRGAIEWEGIESG, and that of peptide R2G is HGGAIEWEGIESG.

Isolation of naive or memory CD4 transgenic T cells

Twelve- to 14-wk-old D10 TCR-Tg or L51S TCR-Tg mice were sacrificed, and their spleens and LN were removed. Single-cell suspensions were prepared and enriched for CD4 T lymphocytes by magnetic bead depletion after staining with mAbs TIB-146 or TIB-164 (anti-B220), TIB-210 or TIB-105 (anti-CD8), 2.4-G2 (anti-FcR), and 10.2.16 (anti-I-Ak), followed by anti-mouse IgG-, anti-rat IgG-, and anti-rat IgM-coated magnetic beads (BioMag, PE Applied Biosystems, Farmingham, MA). The desired CD4 T cells were sorted by FACStar Plus (BD Biosciences, San Jose, CA) after staining with filter-sterilized biotinylated MEL-14 (anti-CD62L) followed by PE-conjugated streptavidin (Caltag, Burlingame, CA) and FITC-conjugated anti-V{alpha}2 TCR (BD PharMingen, San Diego, CA). V{alpha}2+CD62L+ cells were collected and cultured as naive CD4 T cells, and V{alpha}2+CD62L- cells were collected and cultured as the memory phenotype CD4 T cells. For generating effector memory cells we used a previously described protocol with a few modifications (31). D10 TCR-Tg naive CD4 T cells were enriched as described above from the spleens and LN of D10 TCR-Tg mice and were stimulated in vitro with 10 µM CA134–146 peptide in the presence of T-depleted irradiated syngeneic APCs for 72 h. The cells were subjected to Ficoll-Hypaque separation before injection into C{alpha}-/- recipient mice. Cells (107/mouse) were injected into the mice, and the animals were rested for 5–6 wk. Total splenocytes were then collected and stimulated with 10 µM CA 134–146 peptide. Splenic CD4 T cells were analyzed by flow cytometry before peptide stimulation and were fully rested, as indicated by their small size in forward and side scatter. These cells were 100% CD4+3D3+CD44highD10 TCR-Tg memory CD4 T cells. The anti-CD3 mAb used for TCR stimulations was clone 2C11 purified in this laboratory, and it was used at 10 µg/ml in PBS, whereas anti-CD28 mAb was purchased from BD PharMingen and used at 5 µg/ml in PBS.

FACS analysis

Single-cell suspensions from the spleens, LN, and thymi of D10 or L51S TCR-Tg mice were prepared. For splenic and LN suspensions, cells were stained before or after enrichment of CD4 T cells for four-color FACS analysis. All Abs were purchased from BD PharMingen unless otherwise noted. The following Abs were used for staining simultaneously in various combinations as indicated in the figures: biotinylated mAb 3D3 (clonotypic anti-TCR mAb specific to D10 and L51S TCRs) (32), CyChrome-conjugated or PE-streptavidin, FITC-conjugated anti-CD45RB, PE-conjugated anti-CD44, allophycocyanin-conjugated anti-CD62L, biotinylated anti-CD62L (MEL-14; ATCC hybridoma; American Type Culture Collection, Manassas, VA), (allophycocyanin)- and PerCP-conjugated anti-CD4, and FITC-conjugated V{alpha}2. CCR7 was detected using an Fc-CCL19 (ligand for CCR7) extracellular domain fusion protein (gift from J. Cyster (33)), followed by biotinylated anti-human IgG and PE-conjugated streptavidin. Stained cells were analyzed on a FACSCalibur (BD Biosciences), and collected events were analyzed using FlowJo software (Tree Star, San Carlos, CA).

T cell proliferation assays

All T cell proliferation assays were performed in 96-well, U-bottom plates (BD Biosciences, Franklin Lakes, NJ) with 5 x 104 CD4 T cells/well. Total splenocytes from syngeneic nontransgenic C{alpha}-/- littermates were used as a source of APCs after irradiation at 2000 rad. Irradiated splenocytes (1 x 105/well) were added, followed by the addition of varying doses of CA134–145 or R2G peptide ranging from 100–10-3 µM, in a final volume of 200 µl/well. The cultures were left undisturbed for 72 h, at which time each well was pulsed with 0.5 µCi of [3H]thymidine (NEN, Boston, MA). The wells were harvested 18 h later onto printed filter mats (Wallac, Turku, Finland) using a 96-well cell harvester (TomTec, Orange, CT). The filter mats were sealed in sample bags with 5 ml of scintillation fluid (Wallac), and the incorporated radioactivity was counted using a 1205 Betaplate liquid scintillation counter (Wallac, Gaithersburg, MD).

Mini double cultures

These minicultures were adapted from a protocol designed by Dr. K. Bottomly’s research group (34) as a method to provide CD4 T cells with both a primary and a secondary stimulation in vitro. All cultures were performed in duplicate in 96-well, U-bottom plates (BD Biosciences) and in a total volume of 200 µl/well. FACS-sorted D10 TCR or L51S TCR-Tg CD4 T cells (5 x 104) were cultured with 1 x 105 irradiated syngeneic nontransgenic C{alpha}-/- littermate splenocytes and 25 U/ml IL-2 in Click’s modified Eagle’s Ham’s amino acids medium supplemented with 5% FCS (Gemini Bioproducts, Calabasas, CA). CA134–146 or R2G peptide was added to each well at varying doses ranging from 100 to 10-3 µM. Cells were rested on day 4 by removing the entire 200 µl of culture supernatants for cytokine analysis and adding fresh 1 x 105 irradiated syngeneic C{alpha}-/- splenocytes without IL-2 for 48 h. The cultures were then restimulated on day 6 by removing 100 µl of the culture supernatant and replacing it with 100 µl of medium containing 1 x 105 irradiated syngeneic C{alpha}-/- splenocytes to a final concentration of 25 U/ml IL-2 and 100 µM R2G or CA134–146 peptide. Culture supernatants excluding the cells were again removed for cytokine analysis on day 4 after the secondary stimulation.

ELISAs

The levels of IL-4 or IFN-{gamma} in the culture supernatants after primary or secondary stimulation were measured by ELISA. Ninety-six-well Nunc-Immuo plates (Nalge Nunc, Roskilde, Denmark) were coated with 100 µl/well of capturing anti-IL-4 mAb (11B11) and anti-IFN-{gamma} mAb (HB-170) at 6 µg/ml in PBS at 4°C for 18 h. The plates were washed with 0.1% solution of Tween 20 (Sigma-Aldrich, St. Louis, MO) in PBS and blocked with 1% BSA (Sigma-Aldrich) in PBS for 1 h at 37°C. Fifty microliters of culture supernatants and recombinant murine IL-4 or IFN-{gamma} standards (BD PharMingen) were then added and incubated at 4°C for 18 h. Two-fold dilutions of murine recombinant IL-4 or IFN-{gamma} (BD PharMingen) of known concentrations were used as standards. Cytokine-Ab complexes were detected by the addition of biotinylated anti-murine IL-4 or IFN-{gamma} (BD PharMingen), followed by peroxidase-conjugated streptavidin (Zymed Laboratories, San Francisco, CA) and visualization after the addition of o-phenylenediamine dihydrochloride from tablets (Sigma-Aldrich). Color development was stopped with 3 M H2SO4. Absorbance at 492 nm was measured on an ELISA reader (ELx800; Biotek Instruments, Luton, U.K.). The concentrations of IL-4 or IFN-{gamma} in the supernatants were calculated by extrapolating absorbance values from the standard curve, plotting concentration vs absorbance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
An amino acid substitution from L to S at position 51 of the TCR {alpha}-chain results in a memory phenotype in the periphery

We had previously reported that the number of TCR-Tg CD4 T cells in the secondary lymphoid tissues of L51S TCR-Tg mice was markedly reduced (29). The percentage of TCR-Tg T cells in the spleens and LN was detected by staining with the clonotypic D10 TCR-specific mAb 3D3. After CD4 T cell enrichment, 80–90% of the cells were Tg CD4+3D3+ in D10 TCR-Tg mice (Fig. 1A) vs 20–25% in L51S TCR-Tg mice (Fig. 1C). This reduced number of CD4 T cells in the periphery is due to poor positive selection in the thymus (42).



View larger version (63K):
[in this window]
[in a new window]
  		FIGURE 1. L51S TCR-Tg CD4 T cells have a memory phenotype. Enriched CD4 T cells from D10 or L51S TCR-Tg mice were stained and analyzed by FACS. CD4+3D3+ Tg CD4+ T cells were gated (A and C) and their expression of CD44 and CD45RB was analyzed (B and D). Log fluorescence intensity is shown on both the x- and y-axes.

 
During the purification of naive CD4 T cells from L51S TCR-Tg mice we consistently observed a marked reduction in the numbers of naive CD4 T cells compared with CD4 T cells from D10 TCR-Tg mice. To examine this in more detail, we compared L51S TCR-Tg CD4 T cells with D10 TCR-Tg CD4 T cells by FACS analysis for surface naive and memory markers CD44, CD45RB, and CD62L. Naive CD4 T cells were CD44low, CD45RBhigh, and CD62Lhigh, while memory T cells were CD44high, CD45RBneg, and CD62Lneg. TCR-Tg CD4 T cells were selectively examined by staining for CD4 and Tg TCR using the clonotypic mAb 3D3 and gating on CD4+3D3+ double-positive cells (Fig. 1, A and C, for D10 and L51S, respectively). These cells were also simultaneously stained for CD44 and CD45RB. Whereas 92% of D10 TCR-Tg CD4 T cells were CD44lowCD45RBhigh (naive phenotype; Fig. 1B), only 11% of the CD4 T cells from L51S TCR-Tg mice were naive by these same criteria (Fig. 1D). Thus, instead of finding that the majority of CD4 T cells were naïve, as occurs in D10 TCR-Tg mice, 74% of the CD4 T cells from L51S TCR-Tg mice had a memory phenotype, as shown by their surface phenotype, CD45RBlow and CD44high. In contrast, the percentage of memory phenotype D10 TCR-Tg CD4 T cells was only 2.7%. Therefore, the alteration in TCR at position 51 that contacts the {beta}-chain of the MHC class II molecule when the peptide CA134–146 is bound results in the appearance in the periphery of a population of TCR-Tg CD4 T cells that have spontaneously developed a memory cell surface phenotype. The population of CD4+3D3- in Fig. 1C was previously noted (29) and represents V{alpha}2-V{beta}8+ cells. Since these mice were bred on a C{alpha}-/- background, pairing of the V{beta}8 chain with another V{alpha} is impossible, indicating that these cells may express V{beta}8.2 homodimers. The 3D3+ cells that do not express CD4 also do not express CD8, and T cells in this population are also CD44highCD45RBlow. At this time we do not know why these unusual populations appear in L51S mice and are nearly absent from D10 mice (Fig. 1A). Nonetheless, the presence of naive T cells in L51S mice is restricted to the CD4+3D3+ population. Both CD4-3D3+ and CD4+3D3- cells have a memory phenotype, in that they are CD44highCD45RBneg (not shown).

CD44highCD45RBneg L51S TCR-Tg CD4 T cells do not express the LN-homing receptors CCR7 and CD62L

Because we could recover memory phenotype CD44highCD45RBneg L51S TCR-Tg CD4 T cells in the LN as well as the spleen, we further examined the expression of the LN-homing receptors CD62L and CCR7 on these cells. The cells were analyzed by four-color flow cytometry for the simultaneous expression of CD4, the TCR-Tg V{alpha}2 chain, CD62L, and CCR7. The majority (76%) of V{alpha}2-expressing TCR-Tg CD4 T cells shown gated in Fig. 2A are CCR7negCD62Lneg, whereas another 17% were CCR7lowCD62Lneg (Fig. 2B). We compared this expression profile with that of CD4 T cells from young 8-wk-old mice (Fig. 2, C and D) and older 1-year-old mice (Fig. 2, E and F). CD4 T cells from these mice were analyzed by four-color flow cytometry for the simultaneous expression of CD4, CD45RB, CD62L, and CCR7. The naive CD45RBhigh CD4 T cells from young mice shown gated in Fig. 2C were primarily (95%) CCR7+CD62L+ (Fig. 2D). In contrast, the memory CD45RBneg CD4 T cells from older mice shown gated in Fig. 2E were divided into a majority CCR7negCD62Lneg population, a minority CCR7lowCD62Lneg population, and a 10% CCR7lowCD62L+ population. The lack of expression of LN-homing receptors CCR7 and CD62L on L51S TCR-Tg CD4 memory phenotype T cells (Fig. 2B) is very similar to that of memory CD4 T cells in older mice (Fig. 2F). This expression profile is clearly very different from the dual expression of CCR7 and CD62L by naive CD4 T cells (Fig. 2D). In addition, the populations of cells that do express CCR7 in the memory CD4 T cells from older mice (Fig. 2F) and the memory phenotype 3D3+CD4+ T cells from younger L51S TCR-Tg mice (Fig. 2B) are primarily CCR7low.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 2. The majority of L51S TCR-Tg memory CD4 T cells lack expression of CCR7 and CD62L. Enriched CD4 T cells from L51S TCR-Tg or non-Tg mice were stained and analyzed by four-color FACS. A, Gating on V{alpha}2 bearing L51S TCR-Tg CD4 T cells; B, their expression profile of CCR7 and CD62L. C and E, Gating on either CD45RBhigh (naive) or CD45RBneg (memory) CD4 T cells in non-Tg mice; D and F, their corresponding levels of CCR7 and CD62L, respectively. Log fluorescence intensity is shown on both the x- and y-axes.

 
Memory phenotype L51S transgenic CD4 T cells proliferate in response to lower doses of peptide than do naive phenotype L51S transgenic CD4 T cells

To further examine whether the function of memory phenotype L51S TCR-Tg CD4 T cells is reflected by their cell surface phenotype, we isolated both naive and memory phenotype CD4 L51S T cells by sorting with FACS based on their expression of V{alpha}2 and CD62L, with the naive phenotype cells sorted as V{alpha}2+CD62L+ and the memory phenotype cells sorted as V{alpha}2+CD62L- (shown after sorting in Fig. 3, A and B, respectively). Importantly, the bound anti-V{alpha}2 Ab did not result in their nonspecific activation (not shown). The use of anti-V{alpha}2 Ab in sorting this population excluded the populations that were V{beta}8+V{alpha}2-. The proliferation of sorted cells stimulated with the CA-derived peptide CA134–146 and its variant R2G (where R at position 2 of the wild-type peptide was substituted with G) (29, 35) was measured in the presence of irradiated syngeneic I-Ak APC (Fig. 3C). As expected and as previously reported (29, 35), the proliferation of naive L51S CD4 T cells to the R2G mutant peptide occurred at lower doses than that to the CA134–146 peptide. The substitution of R with G at p2 of the peptide shifted the dose-response curve of naive L51S CD4 T cells to 10-fold less peptide. The memory phenotype L51S transgenic CD4 T cells exhibited a similar proliferative response pattern that was also shifted 10-fold compared with that of naive CD4 T cells. In addition, the memory phenotype T cell responses to both CA134–146 and R2G were, on the average, 10-fold more sensitive than the naive T cell responses to the same peptides (Fig. 3C, squares vs circles, respectively).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 3. Memory L51S TCR-Tg CD4 T cells proliferate in response to lower doses of CA134–146 or R2G peptides than their naive counterparts. A and B, Sterile sorting of naive and memory Tg CD4 T cells by FACS based on the expression of the TCR-Tg {alpha}-chain (V{alpha}2+) and CD62L (MEL-14+). Enriched CD4 T cells were stained with anti-V{alpha}2 and biotinylated anti-CD62L, followed by PE-conjugated streptavidin. Naive TCR-Tg V{alpha}2+CD62L+ cells or memory TCR-Tg V{alpha}2+CD62Lneg CD4 T cells were gated and collected. Log fluorescence intensity is shown on both the x- and y-axes. C, Proliferation of sorted naive phenotype and memory phenotype L51S TCR-Tg CD4 T cells. Responses to varying doses of CA134–146 or its related R2G peptide are shown. The x-axis shows the micromolar concentration of peptide. The y-axis shows the counts per minute of [3H]thymidine incorporated.

 
Memory L51S transgenic CD4 T cells express CD25 only after in vitro stimulation

One reason for the enhanced proliferative response of the memory L51S TCR-Tg CD4 T cells may have been that they had already received stimulation in vivo, such that they had increased their levels of the high affinity receptor for IL-2. Fig. 4A shows a flow cytometry profile of these cells immediately following their isolation from the spleens and LN. Gating on the transgenic CD4+3D3+ cells in Fig. 4Aa shows that they express the memory phenotype CD44highCD45RBlow (Fig. 4Ab), and that they are all negative for the expression of CD25 (IL-2R {alpha}-chain; Fig. 4Ac). When CD4+3D3+ T cells from L51S TCR-Tg mice were challenged with a 10-µM dose of CA134–146 peptide in vitro and stained 4 days later, we noted that the 3D3-CD4+ population had disappeared, and 3D3+ cells (both CD4+ and CD4-) dominated the culture (Fig. 4Ba). These cells were again exclusively of the memory phenotype (shown for CD4+ in Fig. 4Bb), but had now increased their expression of CD25 by almost 100-fold (Fig. 4Bc). Based on the lack of CD25 expression ex vivo, the memory phenotype L51S TCR-Tg CD4 T cells most likely did not represent an activated effector population in vivo, and their ability to increase expression of CD25 in vitro was dependent on stimulation with specific Ag.



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 4. L51S TCR-Tg CD4 T cells express CD25 only after in vitro stimulation with specific peptide. A, The staining pattern of enriched CD4 T cells from L51S TCR-Tg mice; B, the staining pattern of CD4 T cells on day 4 after stimulation in vitro with 10 µM CA134–146 peptide. Enriched CD4 T cells were stained and analyzed by FACS. CD4+3D3+ TCR-Tg CD4+ T cells were gated (Aa and Ba), and their expression of CD44 and CD45RB (Ab and Bb) or CD25 and CD45RB (Ac and Bc) was analyzed. Log fluorescence intensity is shown on both the x- and y-axes.

 
Memory phenotype L51S transgenic CD4 T cells lack immediate effector function

One distinguishing functional feature of memory CD4 T cells is that they produce high levels of IL-4 and/or IFN-{gamma} rapidly after only one dose of Ag in vitro, consistent with the idea that their primary antigenic stimulus has already occurred in vivo. Naive T cells, on the other hand, need the first antigenic stimulus in vivo to prime them, followed by a second antigenic stimulus to reveal their true effector and differentiated state. We used an in vitro system of stimulating CD4 T cells that was previously described (29, 34). V{alpha}2+CD62L+ naive phenotype and V{alpha}2+CD62Lneg memoryphenotype TCR-Tg CD4 T cells were isolated from age-matched D10 TCR-Tg and L51S TCR-Tg mice and stimulated with different doses of CA134–146 or R2G peptide. Culture supernatants were collected 3 days after the first peptide stimulation and tested for the presence of IL-4 and IFN-{gamma}. As expected, neither naive D10 TCR-Tg nor naive L51S TCR-Tg CD4 T cells produced IFN-{gamma} after one stimulation with either CA134–146 or R2G (Fig. 5A). IL-4 was also not detected (not shown). In contrast, memory D10 TCR-Tg CD4 T cells produced IFN-{gamma} in a dose-dependent manner in response to both CA134–146 and R2G peptide (Fig. 5A). V{alpha}2+CD62Lneg L51S TCR-Tg CD4 T cells behaved similarly to naive D10 and L51S TCR-Tg CD4 T cells in terms of their cytokine production in response to CA134–146 or R2G. Unlike memory D10 cells, L51S memory phenotype cells did not produce any appreciable IFN-{gamma} or IL-4 (not shown) after one antigenic stimulation in vitro. No cytokines were detected in the absence of peptide from either naive or memory phenotype cell populations.



View larger version (55K):
[in this window]
[in a new window]
  		FIGURE 5. L51S TCR-Tg memory CD4 T cells do not produce IFN-{gamma} after TCR stimulation. A, TCR-Tg CD44lowCD45RBhigh naive or CD44highCD45RBneg memory CD4 T cells from either D10 or L51S mice were cultured in the presence of different doses of CA134–146 or R2G peptide. The primary supernatants were collected on day 4 and tested for the presence of IFN-{gamma} by ELISA. B and C, The cytokine release profile of L51S TCR-Tg CD4 T cells was compared with that of sorted CD44highCD45RBneg D10 TCR-Tg CD4 T cells and that of CA134–146-activated D10 TCR-Tg CD4 T cells that were transferred into C{alpha}-/- recipients, rested 5–6 wk, and isolated as resting memory CD44high3D3+ CD4 T cells. Culture supernatants were tested for the presence of IFN-{gamma} by ELISA. B, IFN-{gamma} release by all groups in response to 10 µM CA134–146 peptide; C, IFN-{gamma} release by all groups after stimulation with anti-CD3 and anti-CD28. C, Culture supernatants were collected at 24, 48, and 72 h as indicated. All IFN-{gamma} release is shown in nanograms per milliliter by 1 x 105 cells/200-µl volume.

 
Since the sorted memory CD4 T cells from older D10 TCR-Tg mice were not bona fide memory CD4 T cells, we used a population of memory D10 TCR-Tg CD4 T cells that were generated in a manner that more closely mimics the physiologic conditions in which memory T cells arise after antigenic challenge in vivo (31). CD4 T cells were isolated from D10 TCR-Tg mice and stimulated with their specific CA134–146 peptide in vitro for 3 days. Subsequently, the cells were transferred into C{alpha}-/- recipient mice and were 100% 3D3+ at the time of transfer. After an in vivo rest period of 5–6 wk the cells recovered were small, fully rested, and 100% 3D3+ and CD44high (not shown). The cells were then stimulated with either specific CA134–146 peptide or anti-TCR mAb plus anti-CD28 mAb to obtain a memory CD4 T cell-characteristic cytokine release profile for comparative purposes. The cytokine release profile of the memory L51S TCR-Tg CD4 T cells was then compared with those of the two different populations of memory D10 TCR-Tg T cells. Fig. 5B shows that when stimulated with 10 µM CA134–146 peptide, L51S TCR-Tg CD4 T cells produced no IFN-{gamma}, whereas D10 TCR-Tg memory T cells and D10 transferred cells produced high levels of IFN-{gamma} (30 and 50 ng/ml, respectively). When stimulated with anti-CD3 and anti-CD28, all cells, including memory phenotype L51S TCR-Tg CD4 cells, produced IL-2 at similar levels (not shown). Both memory populations, D10 and D10 transferred cells, produced high levels of IFN-{gamma}, while L51S memory phenotype cells produced a negligible amount. For both memory D10 TCR-Tg CD4 T cells, the level of IFN-{gamma} measured increased over time (Fig. 5C: 24 h, {square}; 48 h, ; and 72 h, ). In contrast, L51S TCR-Tg memory phenotype CD4 T cells did not secrete IFN-{gamma}. No IL-4 was detected in any of these groups.

Memory L51S TCR-Tg CD4 T cells produce robust levels of effector cytokines only after secondary stimulation

If memory phenotype L51S TCR-Tg CD4 T cells had received a prior TCR stimulation in vivo that resulted in their acquisition of a memory phenotype, we would expect them to execute effector functions as a result of a second antigenic stimulus. Since one restimulation in vitro did not lead to effector cytokine production (Fig. 5), we gave the cells a second in vitro stimulation. In the absence of peptide, both naive and memory phenotype L51S TCR-Tg CD4 T cells failed to survive beyond the primary cultures. This suggested that the memory phenotype L51S cells were not capable of sustained survival in culture in the absence of specific Ag and as such behaved similarly to conventional CD4 T cells. We used two peptides for primary restimulation: the CA134–146 peptide, a weak agonist for the L51S TCR, and the R2G peptide, a strong agonist. A single 10-µM dose of the agonist R2G peptide was used for the second restimulation, which we had previously shown to induce maximal cytokine production (29). We made three observations. First, memory phenotype L51S TCR-Tg CD4 T cells produced robust levels of IL-4 regardless of the nature of the first restimulating peptide (Fig. 6, A and B, for CA134–146 and R2G peptide, respectively). Second, this level of IL-4 was maintained even at low doses (0.001 µM) of restimulating peptide. Third, the dose of peptide used for primary stimulation did not affect the cytokine production profile of resultant effector cells, such that IL-4 was produced at all doses. This was in contrast to the described preferential production of Th2 cytokines at low doses of peptide and Th1 cytokines at high doses of peptide (29, 34, 36). There was a peak of IFN-{gamma} at the 0.1-µM primary stimulating dose of CA134–146 (Fig. 6A) or R2G peptide (Fig. 6B).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 6. L51S TCR-Tg CD4 T cells produce high levels of IL-4 after a second antigenic restimulation. Sorted TCR-Tg V{alpha}2+CD62Lneg memory phenotype (A and B) or V{alpha}2+CD62Lhigh naive phenotype (C and D) L51S TCR-Tg CD4 T cells were cultured in the presence of different doses of CA134–146 (A and C) or R2G peptide (B and D) as shown on the x-axis. After the primary supernatants were collected on day 4 (data shown in Fig. 5, A and B), the cells were restimulated with 10 µM R2G peptide, and the secondary supernatants were collected on day 4. ELISAs were performed in duplicate to measure the concentrations of IL-4 (•) and IFN-{gamma} ({square}). The concentrations of peptide used for the first stimulation are shown on the x-axis. Both IL-4 and IFN-{gamma} release are shown in nanograms per milliliter by 1 x 105 cells/200-µl volume.

 
We compared this cytokine profile to that of naive phenotype L51S TCR-Tg CD4 T cells. As expected and as previously described (29), the differentiation of these naive cells was different depending on the dose and type of priming peptide. Primary stimulation of L51S TCR-Tg CD4 T cells with higher doses (10 µM) of CA134–146 peptide resulted in IL-4-producing cells, a unique property of the altered TCR in these cells, whereas priming with lower doses (1 µM) resulted in IFN-{gamma} production (Fig. 6C). We have previously attributed this reverse cytokine profile to the L51S mutation that lowers the affinity of the TCR to its MHC:CA134–146 peptide ligand (29). When naive L51S TCR-Tg CD4 T cells are primed with the strong agonist peptide R2G, the typical dose-responsive cytokine profile is observed (29, 34, 36). IL-4 production peaked at lower doses of peptide (1 and 0.1 µM), and IFN-{gamma} production peaked at higher doses of peptide (10 µM; Fig. 6D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously described the L51S TCR as a mutant of the D10 TCR that recognizes a CA-derived peptide (CA134–146) presented in the context of I-Ak (29, 30). By destroying the interaction between amino acid 51 in the TCR {alpha}-chain CDR2 region and the highly conserved threonine at position 77 of the MHC class II {beta}-chain, this mutation affects successful contact with position 2 in the peptide ligand. We showed that this mutation dramatically influenced the differentiation path taken by naive TCR-Tg CD4 T cells when primed in vitro with varying doses of CA134–146 peptide (29). The L51S TCR transgenic CD4 T cells differentiated exclusively into Th2-like IL-4 producers at doses of CA134–146 peptide that preferentially primed the parent D10 cells to become Th1-like IFN-{gamma} producers. In the present study as well as that by Dao et al. (29a), we examined several other aspects of the development of these L51S TCR-Tg CD4 T cells.

The L51S mutation results in a 100-fold shift in the peptide dose-response curve when proliferation of L51S TCR-Tg CD4 T cells was measured in response to CA134–146 peptide compared with that of D10 TCR-Tg CD4 T cells (29, 30). In the study by Dao et al. (29a), we show that this reduced proliferation corresponds to a reduced avidity of the L51S TCR for the CA134–146/I-Ak ligand. We do not know what the effect of the L51S mutation is on the avidity of the TCR for selecting ligands in the thymus. In the study by Dao et al. (29a), it was also shown that this single mutation in the TCR {alpha}-chain at a key residue that contacts MHC dramatically alters the numbers of CD4 single-positive mature thymocytes in L51S TCR-Tg mice. The total number of cells in the L51S TCR-Tg thymus is very similar to that in the D10 TCR-Tg thymus. Similarly, the percentage of thymocytes that are double negative is very similar in D10 vs L51S TCR-Tg mice (14%). The only difference in the development of these TCR-Tg thymocytes is the reduced number of CD4 single-positive cells. This difference most likely reflects the inability of the L51S thymocytes to undergo positive selection. It also raises the interesting possibility that the mutation in the L51S TCR not only affects the affinity of the TCR on a mature CD4 T cell for its ligand, but also the affinity of this same TCR on developing thymocytes for their selecting ligand in the thymus.

We noted that the number of naive peripheral L51S TCR-Tg CD4 T cells was markedly reduced compared with that in D10 TCR-Tg mice. Strikingly, the majority (75–90%) of L51S TCR-Tg CD4 T cells had a memory phenotype: CD44highCD45RBnegCCR7neg and L-selectinneg. This was in sharp contrast to D10 TCR-Tg mice as well as to many other previously described TCR-Tg mice, where the vast majority of CD4 T cells are naive. In fact, mice carrying the L51S TCR transgene are the only TCR-Tg mice that have such an abundant memory phenotype population in their secondary lymphoid tissues. These cells possibly acquire the memory phenotype in the periphery upon interactions with either self-peptides or, less likely, peptides derived from environmental Ags. We have, in fact, examined the CD44 and CD45RB expression of mature single-positive CD4 T cells in the L51S thymus that are about to exit to the periphery (unpublished observations). Although the number of cells that we could examine was low due to poor positive selection, the cells appeared to have low levels of CD44 and high levels of CD45RB, suggesting that when exported from the thymus these cells are naive, as would be expected.

We studied the function of memory phenotype L51S TCR-Tg CD4 T cells. We expected that these cells would behave similarly to effector memory T cells based on their phenotype (7). Indeed, these cells proliferated in response to lower doses of antigenic peptide than did naive L51S TCR-Tg CD4 T cells. However, when we examined their ability to rapidly produce high levels of effector cytokines, these cells failed to meet this standard of true effector memory T cell function. When these cells received further TCR stimulation in a culture dish with syngeneic APCs and antigenic peptide, they produced high levels of IL-4 in response to very low doses of peptide Ag, suggesting the ability to further differentiate into effector memory CD4 T cells. This behavior is similar to that described for human TCM cells. Indeed, TCM cells have been suggested to have a precursor relationship to TEM cells. One main difference between L51S TCR-Tg memory CD4 T cells and TCM cells, however, is the lack of CCR7 and CD62L expression, which are considered to be hallmarks of TCM cells based mainly on studies in human T cells (7). It may be that CCR7 and CD62L expression is not a reliable marker for identifying TCM in the mouse. Indeed, one recent study has examined the correlation between CCR7 expression and effector cell function in mice after infection with lymphocytic choriomeningitis virus (LCMV) (37). Both CCR7neg and CCR7+ LCMV-specific TCR-Tg T cells produced IFN-{gamma} after stimulation with Ag and exhibited similar lytic activity toward LCMV peptide-coated target cells. As we show in this study, functional differences between TEM and TCM may be more reliable than the expression of CCR7. Furthermore, the recovery of L51S TCR-Tg memory phenotype CD4 T cells from secondary lymphoid tissues despite the lack of CCR7 and CD62L expression suggests the possible existence of another LN-homing receptor responsible for their distinct homing pattern. It also remains possible that these cells could enter the LN via afferent lymphatics as do true memory cells (3, 5).

The memory phenotype that we describe here for L51S TCR-Tg CD4 T cells is similar to that T cells acquire when adoptively transferred into lymphopenic hosts. This phenotype presumably results from the proliferation that these cells undergo to restore normal cell numbers in the T cell compartment. The cells become CD44highCD45RBneg and L-selectinneg. This proliferation requires interaction with self-peptides presented by MHC class I and MHC class II molecules for CD8 and CD4 T cells, respectively (10, 16, 17, 18, 19, 20). It also requires IL-7/IL-7R interaction (24, 25, 26, 27, 28). The inefficient positive selection that L51S TCR-Tg CD4 T cells undergo in the thymus, as we describe in the accompanying study, may result in a small number of CD4 T cells exiting to the periphery, where they encounter self peptide:self MHC ligands similar to those encountered by adoptively transferred T cells in the earlier homeostasis models (12, 13, 14, 15). The L51S TCR-Tg mice may represent a more physiological mimic of the intentional T cell depletion and irradiation of mice before adoptive T cell transfer in the homeostasis models previously described.

Recently, several studies examined the issue of whether the memory phenotype induced upon homeostasis-driven proliferation of adoptively transferred T cells correlates with memory function (38, 39, 40). These studies were performed on CD8 T cells, and it was concluded that memory phenotype did indeed correlate with function, although this seemed transient and reversible. For example, the memory phenotype enabled the cells to be cytolytic ex vivo, respond to lower doses of Ag than naive T cells, and rapidly produce IFN-{gamma} at early time points after stimulation ex vivo. However, all these new functions disappeared when homeostasis was restored, and the cells reverted to being naive in function while retaining a memory phenotype. In this study we show that the L51S TCR-Tg CD4 T cells have an apparent memory phenotype, but they do not express true effector memory cell function. Thesecells require two rounds of restimulation in vitro to produce effector cytokines such as IL-4 and IFN-{gamma}, suggesting that further differentiation into effector memory CD4 T cells is necessary. Although these cells do not exhibit immediate effector function, they proliferate to in response to lower doses of CA134–146 peptide than their naive counterparts.

There are at least two possible reasons for the appearance of memory phenotype CD4 T cells in L51S TCR-Tg mice. First, and most likely, the cells may proliferate in response to a T cell compartment that is severely underpopulated as a direct result of the poor positive selection observed in the thymus. In this regard, L51S TCR-Tg CD4 T cells may acquire their memory phenotype much like those T cells that undergo homeostatic proliferation when adoptively transferred into T cell-depleted hosts (38, 39, 40). Second, the cells may acquire a memory phenotype because of a possible enhanced reactivity with self peptide:self MHC complexes. This may provide them with a slightly higher than normal level of TCR stimulation by self peptide:self MHC complexes, but not as high as that expected during the course of an immune response to their antigenic ligand. As such, these cells can be placed on the linear differentiation model proposed by Lanzavecchia et al. (7, 41) where naive T cells differentiate first to TCM and then to TEM cells depending on the strength and duration of TCR stimulation. The present study, showing that the presence of memory CD4 T cells in TCR-Tg mice carrying a mutation in the TCR that directly lowers the avidity of the TCR to its ligand, supports the concept that the strength of TCR stimulation directly influences the type of memory T cell generated.

In summary, we describe the L51S TCR Tg mouse, in which small numbers of TCR-Tg CD4 T cells exit the thymus to encounter an environment in the periphery of C{alpha}-/- mice that is devoid of other TCR-bearing CD4 cells. We make two key observations. First, the L51S mutation directly results in the generation of a population of memory phenotype CD4 T cells in the periphery in the absence of immunization. Second, the lack of CCR7 expression by these memory CD4 T cells does not correlate with immediate effector function. Rather, these cells behave similarly to the TCM cells that have been described in humans despite being CCR7neg and CD62Lneg. Memory phenotype L51S TCR-Tg CD4 T cells possess a heightened proliferative response to Ag, but they have not yet acquired the ability to immediately produce the effector cytokines in response to TCR stimulation. Instead, they require further differentiation to do so. We propose that L51S TCR-Tg CD4 T cells have progressed along the differentiation path past the naive stage and toward TEM cells. These cells appear to be at a later differentiation stage than TCM cells, as they have lost the expression of CCR7, a phenotype that is more characteristic of TEM cells. Based on these findings, we propose that L51S memory phenotype CD4 T cells represent a memory CD4 T cell population poised at a stage intermediate between TCM and TEM cells.


   Acknowledgments
 
We thank Tom Taylor for FACS sorting, Gregory Losyev for purification of mAbs, and Charles Annicelli for care of animals.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Charles Janeway, Section of Immunobiology, Yale University School of Medicine, P.O. Box; 208011, New Haven, CT 06520. E-mail address: charles.janeway{at}yale.edu Back

2 Abbreviations used in this paper: HEVs, high endothelial venules; CA, conalbumin; LCMV, lymphocytic choriomeningitis virus; LN, lymph node; TCM, central memory T cell; TEM, effector memory T cell; TCR-Tg, TCR transgenic. Back

Received for publication August 1, 2002. Accepted for publication January 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sprent, J.. 1997. Immunological memory. Curr. Opin. Immunol. 9:371.[Medline]
  2. Tough, D. T., S. Sun, X. Zhang, J. Sprent. 2000. Stimulation of memory T cells by cytokines. Vaccine 18:1642.[Medline]
  3. Swain, S. L., M. Croft, C. Dubey, L. Haynes, P. Rogers, X. Zhang, L. M. Bradley. 1996. From naive to memory T cells. Immunol. Rev. 150:143.[Medline]
  4. Dutton, R. W., L. M. Bradley, S. L. Swain. 1998. T cell memory. Annu. Rev. Immunol. 16:201.[Medline]
  5. Jenkins, M. K., A. Khoruts, E. Ingulli, D. L. Mueller, S. J. McSorley, R. L. Reinhardt, A. Itano, K. A. Pape. 2001. In vivo activation of antigen-specific CD4 T cells. Annnu. Rev. Immunol. 19:23.[Medline]
  6. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101.[Medline]
  7. Sallusto, F., D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708.[Medline]
  8. Wang, X., T. Mosmann. 2001. In vivo priming of CD4 T cells that produce interleukin (IL)-2 but not IL-4 or interferon (IFN)-{gamma}, and can subsequently differentiate into IL-4- or IFN-{gamma}-secreting cells. J. Exp. Med. 194:1069.[Abstract/Free Full Text]
  9. Surth, C. D., J. Sprent. 2000. Homeostatic T cell proliferation: how far can T cells be activated to self-ligands?. J. Exp. Med. 192:F9.
  10. Viret, C., F. S. Wong, C. A. Janeway, Jr.. 1999. Designing and maintaining the mature TCR repertoire: the continuum of self-peptide:self-MHC complex recognition. Immunity 10:559.[Medline]
  11. Goldrath, A. W., M. J. Bevan. 1999. Selecting and maintaining a diverse T-cell repertoire. Nature 402:255.[Medline]
  12. Bell, E. B., S. M. Sparshott, M. T. Drayson, W. L. Ford. 1987. The stable and permanent expansion of functional T lymphocytes in athymic nude rats after a single injection of mature T cells. J. Immunol. 139:1379.[Abstract]
  13. Rocha, B., N. Dautigny, P. Pereira. 1989. Peripheral T lymphocytes: expansion potential and homeostatic regulation of pool sizes and CD4/8 ratios in vivo. Eur. J. Immunol. 19:905.[Medline]
  14. Oehen, S., K. Brduscha-Reim. 1999. Naive cytotoxic T lymphocytes spontaneously acquire effector function in lymphocytopenic recipients: a pitfall for T cell memory studies?. Eur. J. Immunol. 29:608.[Medline]
  15. Beutner, U., H. R. MacDonald. 1998. TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host. Int. Immunol. 10:305.[Abstract/Free Full Text]
  16. Bender, J., T. Mitchell, J. Kappler, P. Marrack. 1999. CD4+ T cell division in irradiated mice requires peptides distinct from those responsible for thymic selection. J. Exp. Med. 190:367.[Abstract/Free Full Text]
  17. Ernst, B., D.-S. Lee, J. M. Chang, J. Sprent, C. D. Surh. 1999. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity 11:173.[Medline]
  18. Goldrath, A. W., M. J. Bevan. 1999. Antagonist ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic hosts. Immunity 11:183.[Medline]
  19. Kieper, W. C., S. C. Jameson. 1999. Homeostatic expansion and phenotypic conversion of naive T cells in response to self peptide/MHC ligands. Proc. Natl. Acad. Sci. USA 96:13306.[Abstract/Free Full Text]
  20. Muranski, P., B. Chmielowski, B., and L. Ignatowicz. 2000. Mature CD4+ T cells perceive a positively selecting class II MHC/peptide complex in the periphery. J. Immunol. 164:3087.
  21. Fung-Leung, W.-P., C. D. Surh, M. Liljedahl, J. Pang, D. Leturcq, P. A. Peterson, S. R. Webb, L. Karlsson. 1996. Antigen presentation and T cell development in H2-M-deficient mice. Science 271:1278.[Abstract]
  22. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois, H. Ploegh, C. Benoist, D. Mathis. 1996. Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84:531.[Medline]
  23. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, L. Van Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection. Cell 84:543.[Medline]
  24. Tan, J. T., E. Dudl, E. LeRoy, R. Murray, J. Sprent, K. I. Weinberg, C. D. Surh. 2001. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc. Natl. Acad. Sci. USA 98:8732.[Abstract/Free Full Text]
  25. Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1:426.[Medline]
  26. Vivien, L., C. Benoist, D. Mathis. 2001. T lymphocytes need IL-7 but not IL-4 or IL-6 to survive in vivo. Int. Immunol. 13:763.[Abstract/Free Full Text]
  27. Goldrath, A. W., P. V. Sivakumar, M. Glaccum, M. K. Kennedy, M. J. Bevan, C. Benoist, D. Mathis, E. A. Butz. 2002. Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8+ T cells. J. Exp. Med. 195:1515.[Abstract/Free Full Text]
  28. Tan, J. T., B. Ernst, W. C. Kieper, E. LeRoy, J. Sprent, C. D. Surh. 2002. Interleukin (IL)-15 and IL-7 jointly regulate homeostatic proliferation of memory phenotype CD8+ cells but are not required for memory phenotype CD4+ cells. J. Exp. Med. 195:1523.[Abstract/Free Full Text]
  29. Blander, J. M., D. B. Sant’Angelo, K. Bottomly, C. A. Janeway, Jr.. 2000. Alteration at a single amino acid residue in the T cell receptor {alpha} chain complementarity determining region 2 changes the differentiation of naive CD4 T cells in response to antigen from T helper type 1 (Th1) to Th2. J. Exp. Med. 191:2065.[Abstract/Free Full Text]
  30. Sant’Angelo, D. B., G. Waterbury, P. Preston-Hurlburt, S. T. Soon, R. Medzhitov, S.-C. Hong, C. A. Janeway, Jr.. 1996. The specificity and orientation of a TCR to its peptide-MHC class II ligands. Immunity 4:367.[Medline]
  31. Rogers, P. R., C. Dubey, S. L. Swain. 2000. Qualitative changes accompany memory T cell generation: faster, more effective responses at lower doses of antigen. J. Immunol. 164:2338.[Abstract/Free Full Text]
  32. Kaye, J., S. Porcelli, J. Tite, B. Jones, C. A. Janeway, Jr.. 1983. Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen presenting cells in the activation of T cells. J. Exp. Med. 158:836.[Abstract/Free Full Text]
  33. Hargreaves, D. C., P. L. Hyman, T. T. Lu, V. N. Ngo, A. Bidgol, G. Suzuki, Y. R. Zou, D. R. Littman, J. G. Cyster. 2001. A coordinated change in chemokine responsiveness guides plasma cell movements. J. Exp. Med. 194:45.[Abstract/Free Full Text]
  34. Pfeiffer, C., J. Murray, J. Madri, K. Bottomly. 1991. Selective activation of Th1- and Th2-like cells in vivo-response to human collagen IV. Immunol. Rev. 123:65.[Medline]
  35. Hong, S. C., D. B. Sant’Angelo, B. N. Dittel, R. Medzhitov, S. T. Yoon, P. G. Waterbury, C. A. Janeway, Jr.. 1997. The orientation of a T cell receptor to its MHC class II:peptide ligands. J. Immunol. 159:4395.[Abstract]
  36. Constant, S., C. Pfeiffer, A. Woodard, T. Pasqualini, K. Bottomly. 1995. Extent of TCR ligation can determine the functional differentiation of naive CD4+ T cells. J. Exp. Med. 182:1591.[Abstract/Free Full Text]
  37. Unsoeld, H., S. Krautwald, D. Voehringer, U. Kunzendorf, H. Pircher. 2002. Cutting edge: CCR7+ and CCR7- memory T cells do not differ in immediate effector cell function. J. Immunol. 169:638.[Abstract/Free Full Text]
  38. Goldrath, A. W., L. Y. Bogatzki, M. J. Bevan. 2000. Naive T cells transiently acquire a memory-like phenotype during homeostasis-driven proliferation. J. Exp. Med. 192:557.[Abstract/Free Full Text]
  39. Cho, B. K., V. P. Rao, Q. Ge, H. N. Eisen, J. Chen. 2000. Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells. J. Exp. Med. 192:549.[Abstract/Free Full Text]
  40. Murali-Krishna, K., R. Ahmed. 2000. Naive T cells masquerading as memory cells. J. Immunol. 165:1733.[Abstract/Free Full Text]
  41. Sallusto, F., D. Lenig, C. R. Mackay, A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875.[Abstract/Free Full Text]
  42. Dao, T., J. M. Blander, D. B. Sant’Angelo. 2003. Recognition of a specific self-peptide: self MHC class II complex is critical for positive selection of thymocytes expressing the D10 TCR. J. Immunol. 170:48.[Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2003 170: 2795-2796. [Full Text]  



This article has been cited by other articles:


Home page
 J. Immunol.Home page
R. Penitente, C. Nicolo, P. Van den Elzen, G. Di Sante, C. Agrati, F. Aloisi, E. E. Sercarz, and F. Ria
Administration of PLP139-151 Primes T Cells Distinct from Those Spontaneously Responsive In Vitro to This Antigen
J. Immunol., May 15, 2008; 180(10): 6611 - 6622.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. J. Maglione, J. Xu, A. Casadevall, and J. Chan
Fc{gamma} Receptors Regulate Immune Activation and Susceptibility during Mycobacterium tuberculosis Infection
J. Immunol., March 1, 2008; 180(5): 3329 - 3338.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Nitcheu-Tefit, M.-S. Dai, R. J. Critchley-Thorne, F. Ramirez-Jimenez, M. Xu, S. Conchon, N. Ferry, H. J. Stauss, and G. Vassaux
Listeriolysin O Expressed in a Bacterial Vaccine Suppresses CD4+CD25high Regulatory T Cell Function In Vivo
J. Immunol., August 1, 2007; 179(3): 1532 - 1541.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
C. Nicolo, G. Di Sante, M. Orsini, S. Rolla, S. Columba-Cabezas, V. R. Spica, G. Ricciardi, B. M. C. Chan, and F. Ria
Mycobacterium tuberculosis in the adjuvant modulates the balance of Th immune response to self-antigen of the CNS without influencing a "core" repertoire of specific T cells
Int. Immunol., February 1, 2006; 18(2): 363 - 374.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
F. S. Dhabhar and K. Viswanathan
Short-term stress experienced at time of immunization induces a long-lasting increase in immunologic memory
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2005; 289(3): R738 - R744.